The long-range goal of this project is to identify mechanisms regulating synaptic connectivity in the mammalian neocortex and to elucidate how their deficiency causes abnormal brain wiring relevant to neurodevelopmental disorders such as autism spectrum disorders (ASDs). This application seeks to elucidate a novel mechanism for spine and synapse regulation involving NrCAM (Neuron-Glial Related Cell Adhesion Molecule), a risk factor in ASD, which is important for development of excitatory circuits in the neocortex. A new concept to be studied is that NrCAM regulates spine development by interacting with repellent guidance molecules Semaphorin3F (Sema3F), Neuropilin-2 (Npn-2), and PlexinA3 (PlexA3), and that NrCAM deficiency leads to hyperexcitability in neocortical circuits. The central hypothesis to be investigated is that NrCAM/Npn- 2/PlexA3 comprises a receptor complex for Sema3F that constrains or remodels dendritic spines of pyramidal neurons in the neocortex for appropriate excitatory balance. Aims to be addressed are as follows: Aim 1: A role for NrCAM in constraining spine and excitatory synapse formation will be investigated by analyzing dendritic spines and synapses of pyramidal neurons in prefrontal and sensory cortical areas of wild type (WT) and NrCAM null mice, and localizing NrCAM to synaptic sites. A double heterozygote analysis will be undertaken to investigate the postulated genetic interaction of NrCAM with Npn-2, PlexA3, and Sema3F to regulate spine morphogenesis in vivo. Aim 2: To determine if NrCAM loss induces hyperexcitability of pyramidal neurons, excitatory responses will be measured in star pyramidal neurons, the principal target of thalamocortical input to visual cortex (layer 4), in visual cortical slices of WT and NrCAM null mutant mice by whole cell recordings, and the effect of Sema3F treatment on excitatory responses in WT and null mutant slices will be compared. Aim 3: A cell autonomous, postsynaptic mechanism for NrCAM will be investigated for Sema3F-induced spine morphogenesis mediated by interaction with Npn-2 and PDZ adaptors. Genetic rescue of spine morphogenesis will be analyzed in star pyramidal neurons of NrCAM null embryos electroporated in utero with WT and NrCAM binding mutants for Npn-2 and PDZ adaptors, and in Sema3F-treated neuronal cultures. The outcome of these studies is expected to have an important positive impact, because it will delineate novel molecular mechanisms of spine morphogenesis that control sensory cortical connectivity and function, and may provide insight into molecular mechanisms targeted in ASDs. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health, because it seeks to define molecular determinants that regulate development of neural connectivity in the mammalian brain. The research is relevant to the mission of NIH in illuminating basic mechanisms of neurodevelopment important for understanding inherited brain disorders, and especially relevant to sensory abnormalities in autism and related syndromes, termed Autism Spectrum Disorders, for which NrCAM, Neuropilin-2, and Semaphorins are candidate risk factors.